Natural yeast and lactic acid bacteria isolated from korean traditional nuruk to be used for bakery

ABSTRACT

Provided are  Saccharomyces cerevisiae  SPC-SNU 70-1 (KCTC 12776BP) which is novel natural Korean lactic acid bacteria isolated from traditional Korean nuruk, and  Lactobacillus brevis  SPC-SNU 70-2 (KCTC 12777BP),  Lactobacillus curvatus  SPC-SNU 70-3 (KCTC 12778BP) and  Lactobacillus sanfranciscensis  SPC-SNU 70-4 (KCTC 12779BP) which are novel natural Korean yeasts isolated from traditional Korean nuruk.

TECHNICAL FIELD

The present invention relates to novel natural Korean yeast and natural Korean lactic acid bacteria for bread making (baking) isolated from traditional Korean Nuruk and more specifically, to Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) which is a novel natural Korean lactic acid bacteria isolated from traditional Korean nuruk, and Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP), Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) which are novel natural Korean yeasts isolated from traditional Korean nuruk.

BACKGROUND ART

Sourdough is also called “acid dough” and has unique flavor and taste. Fermentation using sourdough generally includes repeating a series of mixing flour, water and rye to activate microorganisms present therein, thereby preparing a sourdough starter, and using some sourdough starter for dough and storing the remaining for the subsequent use. Fermentation using sourdough includes a fermentation process in which yeast and lactic acid bacteria are mutually involved, and have several advantages associated with baking properties such as increased bread volume, improved flavor and taste, and extended expiration date.

Meanwhile, microorganisms greatly affect fermentation of sourdough. In particular, it is known that main fermentation microorganisms, i.e., lactic acid bacteria and yeast, act on fermentation alone or in combination thereof to make sourdough. The main cause of sour taste is lactic acid produced from lactic acid bacteria, which is a base of sourdough, and alcohol fermentation metabolites generated by yeast impart taste to bread and improve palatability. The genera Streptococcus, Pediococcus, Lactobacillus, Enterococcus, Leuconostoc, Weissella and the like are known lactic acid bacteria related to fermentation of sourdough.

Meanwhile, traditional sourdough bread produced by natural fermentation is mixed with a variety of yeasts and bacteria. For this reason, problems such as deterioration in flavor and taste and creation of rancid odor often result from contamination by other microorganisms under inappropriate work environments. In addition, there is difficulty in reproducing uniform quality of products due to spatial and timing differences.

In an attempt to solve these problems, in order to avoid the risk of contaminating naturally fermented bread and secure uniform quality of products, fermentation including adding microorganism starters with secured functionality and stability to dough was developed.

Some Korean baking companies use imported starters to pursue differentiation and high-quality of products. However, imported starters change strain distribution during subculture, are easy to handle in industrial bakeries and have many practical problems such as difficulty in controlling sour taste levels because they contain, as dominant species, L. sanfranciscensis strains producing a great amount of organic acid. In addition, imported starters may have quality properties unsuitable for domestic baking circumstances because they are selected in consideration of climate and baking properties of countries of origin. Accordingly, there is a need for development of starters which are suitable for Korean tastes and can be stably used in baking fields.

DISCLOSURE Technical Problem

Therefore, it is one object of the present invention to provide novel natural Korean yeast and lactic acid bacteria isolated from traditional Korean Nuruk.

Technical Solution

In accordance with a first aspect of the present invention, the above and other objects can be accomplished by the provision of Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP). The Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) is novel Korean yeast isolated from Nuruk, which is grown well in flour dough with low pH owing to high acid resistance.

Meanwhile, in a second aspect of the present invention, provided is dough for baking produced by adding Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) to flour, followed by fermentation. The flour dough containing Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP), which is novel Korean yeast isolated from traditional Korean Nuruk, has better gas generation capability and is thus suitable for baking, as compared to flour dough containing commercial yeast.

In the second aspect of the present invention, the dough for baking is preferably produced by further adding one or more selected from Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP), Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) to flour, followed by fermentation.

Meanwhile, in the production of sourdough, when lactic acid bacteria is excessively proliferated, lactic acid bacteria produces excess lactic acid to render strong acid taste and uses nutrients necessary for yeast bacteria, which inhibits growth of yeasts. On the other hand, when the number of initial yeasts is excessively high, effect caused by addition of lactic acid bacteria is lowered. Accordingly, it is very important to secure appropriate numbers of living yeasts and lactic acid bacteria during fermentation in order to produce sourdough with excellent tastes. For this purpose, there is a need to use as starters, symbiotically grown yeast and lactic acid bacteria strains, rather than competitively grown yeast and lactic acid bacteria strains.

Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP) and Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP), which are novel Korean yeasts isolated from Nuruk according to the present invention, have high resistance to acid and superior maltose availability and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) produces less acid and has superior maltose availability and thus grows rapidly. In addition, Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP), which is novel Korean yeast isolated from traditional Korean Nuruk, has high resistance to acid. When Korean yeasts and lactic acid bacteria having properties as described above are applied in combination to dough, yeasts and lactic acid bacteria are grown symbiotically, bread having great specific volume, excellent texture, and superior flavor and taste can be produced. That is, when yeasts and strains isolated from traditional Korean Nuruk are used, bread suitable for Korean tastes can be produced as compared to when conventional imported starters are used.

Meanwhile, in a third aspect of the present invention, provided is a bread produced by adding Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) to flour, followed by fermentation and baking. The bread produced by the method has greater specific volume, lower hardness and slower aging rate and thus better storage property than bread produced with commercial yeast. In addition, the bread according to the present invention emits aroma ingredients which are soft and have superior preference, thus exhibiting good flavor and superior preference.

In this aspect, the bread is preferably produced by further adding one or more selected from Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP), Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) to flour, followed by fermentation and baking.

In this aspect, the baking means baking bread.

Effects of the Invention

The present invention provides Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) which is novel natural Korean lactic acid bacteria isolated from traditional Korean nuruk, and Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP), Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) which are novel natural Korean yeasts isolated from traditional Korean nuruk.

The dough for baking using the novel natural Korean yeast and natural Korean lactic acid bacteria secure appropriate numbers of living yeasts and lactic acid bacteria and thus exhibit superior gas generation capability. In addition, bread produced using flour dough containing the novel natural Korean yeast and natural Korean lactic acid bacteria has great specific volume and low hardness, is soft and thus has excellent texture, renders aroma ingredients with excellent preference and is thus suited to Korean tastes.

DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows identification results of microorganisms isolated from Nuruk, flour, natural sourdough and rye;

FIG. 2 shows results of PCR using Lactobacillus sanfranciscensis-specific primers and then electrophoresis conducted on strains isolated from natural sourdough;

FIG. 3 shows results of PCR using Lactobacillus brevis-specific primers and then electrophoresis conducted on strains isolated from natural sourdough;

FIG. 4 shows results of PCR using Saccharomyces cerevisiae-specific primers and then electrophoresis conducted on isolated yeasts;

FIG. 5 is a graph showing comparison in maximum lactic acid production of Lactobacillus sanfranciscensis strains isolated from natural sourdough;

FIG. 6 shows a fermentation profile of Group 1 strains among Lactobacillus sanfranciscensis strains isolated from natural sourdough;

FIG. 7 shows a confirmation result of acid resistance of Lactobacillus brevis isolated from natural sourdough;

FIG. 8 shows a confirmation result of maltose availability of Lactobacillus brevis isolated from natural sourdough;

FIG. 9 shows a confirmation result of acid resistance against a variety of acids with respect to Saccharomyces cerevisiae (S. cerevisiae) 01435 isolated from natural sourdough;

FIG. 10 shows images of control group (commercial yeast-applied bread) and isolated yeast S. cerevisiae 01435-applied bread;

FIG. 11 is a graph showing a gas generation capability of dough containing S. cerevisiae 01435 isolated from natural sourdough;

FIG. 12 shows a confirmation result of aging rate of bread containing S. cerevisiae 01435 isolated from natural sourdough;

FIG. 13 shows a comparison result of quantitative values of aroma ingredients of bread containing S. cerevisiae 01435 isolated from natural sourdough;

FIG. 14 shows images of a control group (only commercial yeast-applied bread), reference (well-known) strain (L. curvatus KCCM40715)-applied bread and isolated strain (L. curvatus 104)-applied bread;

FIG. 15 shows a confirmation result of gas generation capability of dough containing Lactobacillus curvatus (L. curvatus) 104 isolated from natural sourdough;

FIG. 16 shows a confirmation result of aging rate of bread containing Lactobacillus curvatus (L. curvatus) 104 isolated from natural sourdough;

FIG. 17 shows a comparison result of quantitative values of aroma ingredients of bread containing L. curvatus 104 isolated from natural sourdough;

FIG. 18 shows images of control group (only commercial yeast-applied bread), reference strain (L. brevis KACC 11433)-applied bread and isolated strain (L. brevis 149)-applied bread;

FIG. 19 shows a confirmation result of gas generation capability of dough containing Lactobacillus brevis (L. brevis) 149 isolated from natural sourdough;

FIG. 20 shows a confirmation result of aging rate of bread containing L. brevis 149 isolated from natural sourdough;

FIG. 21 shows a comparison result of quantitative values of aroma ingredients of bread containing L. brevis 149 isolated from natural sourdough;

FIG. 22 shows images of control group (only commercial yeast-applied bread), reference strain (L. sanfranciscensis KACC 12431)-applied bread and isolated strain (L. sanfranciscensis 142)-applied bread;

FIG. 23 shows a confirmation result of gas generation capability of dough containing Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 isolated from natural sourdough;

FIG. 24 shows a confirmation result of aging rate of bread containing Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 isolated from natural sourdough;

FIG. 25 shows a comparison result of quantitative values of aroma ingredients of bread containing Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 isolated from natural sourdough;

FIG. 26 shows images of only commercial yeast-applied bread (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 (E set)-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 (F set)-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 (G set)-applied bread, and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104, Lactobacillus brevis 149 and Lactobacillus sanfranciscensis 142 (H set)-applied bread;

FIG. 27 shows confirmation results of gas generation capability of only commercial yeast-applied dough (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied dough (E set-applied dough), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied dough (F set-applied dough), Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied dough (G set-applied dough), and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied dough (H set-applied dough);

FIG. 28 shows confirmation results of aging rates of only commercial yeast-applied bread (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread (E set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread (F set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread (G set-applied bread), and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread (H set-applied bread); and

FIG. 29 is a comparison result of quantitative values of aroma ingredients of only commercial yeast-applied bread (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread (E set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread (F set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread (G set-applied bread), and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread (F set-applied bread).

BEST MODE

Hereinafter, the present invention will be described in more detail with reference to the following examples, and the scope of the present invention is not limited to the examples and includes variations of technical concepts equivalent thereto.

Meanwhile, in the present invention, Saccharomyces cerevisiae (S. cerevisiae) 01435 isolated by the following examination was designated “Saccharomyces cerevisiae SPC-SNU 70-1” and then deposited under the accession number “KCTC 12776BP”. In addition, a Lactobacillus curvatus (L. curvatus) 104 strain was designated “Lactobacillus curvatus SPC-SNU 70-3” and then deposited under the accession number “KCTC 12778BP”. In addition, a Lactobacillus brevis (L. brevis) 149 strain was designated “Lactobacillus brevis SPC-SNU 70-2” and then deposited under the accession number “KCTC 12777BP”. In addition, a Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 strain was designated “Lactobacillus sanfranciscensis SPC-SNU 70-4” and then deposited under the accession number “KCTC 12779BP”.

Preparation Example 1 Preparation of Nuruk-Containing Natural Sourdough (Nuruk-Containing Korean Sourdough)

In order to extract microorganisms from Nuruk, 450 g of feed water was added to 50 g of Nuruk, cultured in an incubator at a temperature of 22° C. and at a humidity of 83% for 4 hours and bran of Nuruk was removed using a 100 mesh sieve to prepare a starter filtrate.

In a first step of preparing a natural sourdough, 400 g of the starter filtrate, 950 g of flour and 100 g of a rye powder were homogenously mixed with 650 g of room-temperature heating/cooling water and a starter was cultured by fermentation at a temperature of 25° C. and at a humidity of 85% for 48 hours to prepare a starter culture.

In a second step of preparing natural sourdough (preparation of natural sourdough and subculture), 700 g of the prepared starter culture was homogenously mixed with 1,050 g of heating/cooling water, 950 g of flour and 100 g of a rye powder to adjust a temperature of the kneaded substance to 26° C., and the mixture was fermented in an incubator at a temperature of 12° C. for 15 hours to prepare a natural sourdough. The storage was conducted in a refrigerator at 2 to 4° C. For subculture, 700 g of the natural sourdough stored in the refrigerator was homogeneously mixed with 1,050 g of the heating/cooling water, 950 g of flour and 100 g of rye to adjust a temperature of the kneaded substance to 26° C., and the mixture was fermented in an incubator at a temperature of 12° C. for 15 hours to prepare a natural sourdough. The series of steps were repeated to complete subculture.

Example 1 Isolation and Identification of Microorganisms

Microorganisms were isolated from Nuruk, rye, flour and the natural sourdough (Preparation Example 1). 10 g of each raw material and 90 mL of 0.85% NaCl were put into a filter bag and these ingredients were homogenized using a stomacher for 3 minutes. The resulting mixture was diluted to an appropriate concentration by stepwise dilution using 0.85% NaCl, lactic acid bacteria was smeared on MRS (de Man Rogosa and Sharpe, Difco) supplemented with 0.01% cycloheximide and SDB (2% maltose, 0.3% yeast extract, 1.5% fresh yeast extract, 0.03% Tween 80, 0.6% casein peptone, pH 5.6) solid media and isolated. Yeast was smeared on 0.35% sodium propionate-containing YM (yeast malt extract) and PDA (potato dextrose agar) solid media and isolated.

The MRS was subjected to stationary culture at 37° C. and SDB, YM and PDA media were subjected to stationary culture at 30° C. Then, to obtain single colonies, respective colonies were sub-cultured 3 to 4 times and subjected to cytomorphological examination and gram staining, and bacteria (lactic acid bacteria) were identified by 16s rRNA sequence analysis, and yeasts were identified by ITS sequence analysis.

As a result, lactobacillus curvatus (L. curvatus), Pediococcus pentosaceus (P. pentosaceus), lactobacillus sakei (L. sakei), lactobacillus plantarum (L. plantarum) and Pediococcus acidilactici (P. acidilactici) were isolated as lactic acid bacteria from nuruk, and Torulaspora delbrueckii (T. delbrueckii), Pichia anomala (P. anomala), Saccharomyces cerevisiae (S. cerevisiae), Candida krusei (C. krusei) and Candida pelliculosa (C. pelliculosa) were isolated as yeasts from Nuruk.

In addition, Pediococcus pentosaceus (P. pentosaceus), Lactobacillus brevis (L. brevis), Lactobacillus plantarum (L. plantarum), Lactobacillus curvatus (L. curvatus), Lactobacillus sakei (L. sakei), Lactobacillus crustorum (L. crustorum), and Lactobacillus sanfranciscensis (L. sanfranciscensis) were isolated as lactic acid bacteria from the natural sourdough and Saccharomyces cerevisiae (S. cerevisiae), Candida krusei (C. krusei) and Candida pelliculosa (C. pelliculosa) were isolated as yeasts from the natural sourdough.

In addition, Pediococcus pentosaceus (P. pentosaceus) was isolated from flour and Pediococcus pentosaceus (P. pentosaceus) and Lactobacillus curvatus (L. curvatus) were isolated from rye.

FIG. 1 shows identification results of microorganisms isolated from Nuruk, flour, natural sourdough and rye.

Example 2 Isolation of Lactobacillus sanfranciscensis (L. sanfranciscensis) from Natural Sourdough

In the present example, Lactobacillus sanfranciscensis (L. sanfranciscensis) was isolated from natural sourdough (Nuruk-containing Korean sourdough). For this purpose, L. sanfranciscensis-specific PCR was designed.

The whole genomic sequence of L. sanfranciscensis was found in 2011 (Rudi F Vogel, Melanie Pavlovic, Matthias A Ehrmannl, Arnim Wiezer, Heiko Liesegang, Stefanie Offschanka, Sonja Voget, Angel Angelov, Georg BoWolfgang Liebl (2011) Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs. Microbial Cell Factories. 10(Suppl 1):S6), and genes which do not overlap other strains were searched in the genomic map and gene database of Lactobacillus sanfranciscensis TMW 1.1304 using the same, and among the genes, LSA_02510 hypothetical protein was selected.

PCR primers were designed such that they are not amplified in genes of other strains, and PCR was conducted using the primer sanhypl: 5′GGAGGAAA ACTCATGAGTGTTAAG3′(24mer) and the primer sanhyp2: 5′CAAAGTCA-AGAAGTTATCCATAAACAC (27mer) under the following conditions: pre-denaturation at 94° C. for 5 minutes; 30 cycles at 94° C. for 30 seconds, at 63° C. for 30 seconds and at 72° C. for 1 minute; and final extension at 72° C. for 7 minutes.

Genomic DNAs were isolated from 68 single colonies isolated from natural sourdough, Lactobacillus sanfranciscensis-specific PCR was conducted using the same as templates, and electrophoresis was performed.

As a result, among 68 samples, 39 samples exhibited 957 bp bands whereas the remaining samples exhibited no band. All 39 samples exhibiting bands were subjected to 16s rRNA sequence analysis. As a result, the samples were identified as Lactobacillus sanfranciscensis.

FIG. 2 shows results of PCR using Lactobacillus sanfranciscensis-specific primers and then electrophoresis conducted on strains isolated from natural sourdough.

Example 3 Isolation of Lactobacillus curvatus (L. curvatus) from Natural Sourdough—L. curvatus-Specific PCR

In the present example, Lactobacillus curvatus-specific PCR was designed to isolate Lactobacillus curvatus (L. curvatus) from natural sourdough (Nuruk-containing Korean sourdough).

Genes having almost no homogeneity to other lactic acid bacteria were searched by NCBI Blast search and “CRL 705 contig 00107” was selected. PCR primers were designed such that they were not amplified in genes of other strains, PCR was conducted using the primers F′-CUR 5′-GACCCATGCCTTT AATACGCATAG-3′ and R′-CUR 5′-CTGAAATAACCACTATAGCCACCCC-3′ under the following conditions: pre-denaturation at 94° C. for 5 minutes; 40 cycles at 94° C. for 30 seconds, at 61.5° C. for 30 seconds and at 72° C. for 1 minute; and final extension at 72° C. for 7 minutes.

Meanwhile, genomic DNAs were isolated from 68 single colonies isolated from natural sourdough, L. curvatus-specific PCR was conducted using the same as templates, and electrophoresis was performed.

As a result, 35 samples exhibited 129 bp bands whereas the remaining samples exhibited no band. All 35 samples exhibiting bands were subjected to 16s rRNA sequence analysis. As a result, the samples were identified as Lactobacillus curvatus.

Example 4 Isolation of Lactobacillus brevis (L. brevis) from Natural Sourdough—L. brevis-Specific PCR

In the present example, Lactobacillus brevis-specific PCR was designed to isolate Lactobacillus brevis (L. brevis) from natural sourdough (Nuruk-containing Korean sourdough).

(1) Production of Selection Medium and Culture Conditions

mMRS-BPB selection media was used to isolate Lactobacillus brevis (L. brevis) from natural sourdough (Nuruk-containing Korean sourdough). mMRS-BPB was obtained by adding 0.2 g/400 mL of cysteine-HCl and 8 mg/400 mL of bromophenol-blue to a conventional MRS medium, autoclaving the medium at a temperature of 121° C. for 15 minutes and adding 20 mg/mL D.W of filtered cycloheximide thereto on the basis of 2 mL/400 mL. Bromophenol-blue was added to selectively choose strains using its property in which colony changes color depending on acid produced by lactic acid bacteria, and cycloheximide was used to inhibit fungal growth. Culturing was carried out by anaerobic culturing at 37° C. for 24 hours.

(2) Isolation of Lactic Acid Bacteria

5 g of natural sourdough was mixed with 45 mL of 0.85% NaCl, and the mixture was homogenized using a stomacher, was diluted to 1×10⁻⁶ using 0.85% NaCl and was smeared on an amount of 100 μl in a mMRS-BPB medium. The resulting substance was subjected to anaerobic culture at 37° C. for 24 hours, and plain pattern pale sky-blue colonies were considered Lactobacillus brevis (L. brevis) candidate strains and subjected to pure culture.

(3) Isolation of Lactobacillus brevis (L. brevis)—L. brevis Specific PCR

Primers specific to Lactobacillus brevis (L. brevis) used herein were BRE-F (5′-CAGTTAACTTTTGCGAGTCAGCAG-3′) and BRE-R (5′-CGTCAGGTTCCCCACATAACTC-3′) designed with reference to the research by Hyun-wook Baek et al.(Hyun-wook Baek, 2014, Thesis, SNU, Investigation of microbial diversity in Korean sourdough and its monitoring by real-time quantitative PCR), an amplification size was 162 bp, colony PCR was conducted under the following conditions: pre-denaturation at 95° C. for 10 minute; 30 cycles at 95° C. for 30 seconds, at 61.5° C. for 30 seconds and at 72° C. for 20 seconds; and final extension at 72° C. for 20 seconds, and electrophoresis was conducted.

As a result, 7 samples exhibited 162 bp bands. All samples exhibiting bands were identified to be Lactobacillus brevis (L. brevis).

FIG. 3 shows results of PCR using Lactobacillus brevis-specific primers and then electrophoresis conducted on strains isolated from natural sourdough.

Example 5 Isolation of Saccharomyces cerevisiae (S. cerevisiae) from Natural Sourdough and Identification

In the present example, Saccharomyces cerevisiae-specific PCR was designed to isolate Saccharomyces cerevisiae (S. cerevisiae) from natural sourdough.

(1) Yeast Isolation and Culture Conditions

In order to isolate yeasts from early natural sourdough, mature natural sourdough (stabilized state), Panetone sourdough, and Sanfrancisco sourdough, 10 g of each raw material and 90 mL of 0.85% NaCl were put into a filter bag and these ingredients were homogenized using a stomacher for 3 minutes. The resulting mixture was diluted to an appropriate concentration by stepwise dilution using 0.85% NaCl, and smeared on an YPM (1% yeast extract, 2% peptone, 2% maltose) solid medium supplemented with 0.35% sodium propionate to specifically isolate yeasts. Culturing was carried out by stationary culturing at 30° C. Then, in order to obtain single colonies, respective colonies were sub-cultured 3 to 4 times, and Saccharomyces cerevisiae (S. cerevisiae) candidate strains were selected from the isolated single colonies using Saccharomyces cerevisiae (S. cerevisiae)-specific primers.

(2) Selection of Saccharomyces cerevisiae (S. cerevisiae)—S. cerevisiae-Specific PCR

The Saccharomyces cerevisiae (S. cerevisiae)-specific primers used herein were SCDF (5′-AGG AGT GCG GTT CTT TG-3′) and SCDR (5′-TAC TTA CCG AGG CAA GCT ACA-3′) designed with reference to the research by Ho-Won Chang et al. (Ho-Won Chang, Young-Do Nam, Youlboong Sung, Kyoung-Ho Kim, Seong Woon Roh, Jung-Hoon Yoon, Kwang-Guk An, Jin-Woo Bae, 2007, Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces, Journal of Microbiological Methods, Vol. 71, Issue 3). The primers were reported as specific primers which set D1/D2 regions of Saccharomyces cerevisiae to a size of 310 bp and do not amplify genes of other yeasts.

The Saccharomyces cerevisiae candidate strains isolated in (1) were subjected to colony PCR using SCDF and SCDR primers under the following conditions: pre-denaturation at 94° C. for 5 minutes; 30 cycles at 94° C. for 1 minute, at 60° C. for 1 minute and at 72° C. for 1 minute; and final extension at 72° C. for 7 minutes, and then to electrophoresis.

As a result of testing, as can be seen from FIG. 4, among 45 microorganisms isolated from early natural sourdough, mature natural sourdough (stabilized state), Panetone sourdough and Sanfrancisco sourdough, 36 samples exhibited bands at 310 bp. FIG. 4 shows results of PCR using Saccharomyces cerevisiae-specific primers and then electrophoresis conducted on isolated yeasts. In this case, some of Panetone sourdough and Sanfrancisco sourdough samples exhibited no band amplification.

Example 6 Selection of Good Strains as Starters from Lactobacillus sanfranciscensis (L. sanfranciscensis) Isolated from Natural Sourdough—Confirmation of Acid Production Level and Maltose Availability

In the present example, in order to select good strains acting as starters from Lactobacillus sanfranciscensis (L. sanfranciscensis) isolated from natural sourdough, acid production capability and maltose availability of L. sanfranciscensis strains were compared.

The strain stored at −80° C. was pre-cultured in 10 mL mMRS (based on 1 L, polypeptone 10 g, meat extract 10 g, yeast extract 5 g, Tween80 1 mL, K₂HPO₄ 2 g, sodium acetate 5 g, ammonium citrate 2 g, MgSO₄ 0.2 g, MnSO₄ 0.05 g, maltose or glucose 20 g, pH 5.4, sugar separately autoclaved) broth at a temperature of 30° C. for 24 hours. The cultured cells were collected by centrifugation (4° C., 10,000 rpm), were inoculated in 100 mL of mMRS broth at an early O.D. (at 600 nm) of 0.1, and cultured at 30° C. and at 90 rpm, and the sample was collected at intervals of 3 to 4 hours.

Organic acid was measured using HPLC (Agilent 1100 series, USA). Regarding HPLC conditions, a 0.001N sulfuric acid solution was flowed as a mobile phase at a flow rate of 0.6 mL/min, and a column for analyzing organic acid heated to 60° C. (Rezex ROA-organic acid, Phenomenex, USA) and an RI detector were used.

As a result, it was confirmed that top six strains which produced less lactic acid were ss 131, ss 136, ss 135, ss 142 and ss 161 (Group 1), and the next top five strains were ss 204, ss 194, ss 121, ss 205, and ss 223 (Group 2) (FIG. 5). FIG. 5 is a graph showing comparison in maximum lactic acid production of Lactobacillus sanfranciscensis strains isolated from natural sourdough.

Strains which produced as little lactic acid as possible and had the most potent metabolic capability with respect to maltose were selected by comparing in characteristics between strains of Group 1.

The result of selection showed that the strain ss 142 completely metabolized maltose and produced less lactic acid (FIG. 6). The result indicated that the strain ss 142 had the most suitable properties as a starter. FIG. 6 shows a fermentation profile of Group 1 strains among Lactobacillus sanfranciscensis strains isolated from natural sourdough.

Meanwhile, Lactobacillus sanfranciscensis strain ss 142 was designated “Lactobacillus sanfranciscensis SPC-SNU 70-4” and was deposited under the accession number “KCTC 12779BP”.

Example 7 Selection of Good Strain as Starter from Lactobacillus curvatus (L. curvatus) Isolated from Natural Sourdough—Confirmation of Acid Resistance at Different pH Levels and Maltose Availability

In the present example, acid resistance at different pH levels and maltose availability were confirmed for lactobacillus curvatus strains in order to select superior strains as starters from Lactobacillus curvatus (L. curvatus) isolated from natural sourdough.

(1) Confirmation of Acid Resistance at pH Levels with Respect to Lactobacillus curvatus (L. curvatus) Isolated from Natural Sourdough.

In order to confirm acid resistance of Lactobacillus curvatus 35 species of strains (100, 104, 106, 109, 112, 114C, 114SS, 116, 120, 122, 127, 130, 132, 134, 138, 140N, 140SS, 144, 146, 156N, 156SS, 159, 171N, 172, 183N, 206N, 206SS, 216N, 238, 241N, 241SS, 249N, 250N, 253SS and 253N) isolated from natural sourdough, MRS broth with controlled pH levels of 6.8, 4.6, 4.4 and 4.2 was inoculated with 1% of each of the strains, absorbance was measured at 600 nm after 24 hours and a growth curve was drawn.

As a result, it could be confirmed that strains which had high absorbance at pH 4.2 were 249N, 250N, 206SS and 104 which had absorbance of 1.540, 1.501, 1.446, and 1.403, respectively. In addition, the differences in absorbance between the reference (well-known) strain (L. curvatus KCCM 40715) and the respective strains were 0.207, 0.168, 0.113 and 0.070.

(2) Confirmation of Maltose Availability of Lactobacillus curvatus Isolated from Natural Sourdough

In order to confirm maltose availability of 35 Lactobacillus curvatus species (100, 104, 106, 109, 112, 114C, 114SS, 116, 120, 122, 127, 130, 132, 134, 138, 140N, 140SS, 144, 146, 156N, 156SS, 159, 171N, 172, 183N, 206N, 206SS, 216N, 238, 241N, 241SS, 249N, 250N, 253SS, 253N) isolated from natural sourdough, a medium containing 2% maltose instead of dextrose with respect to the composition of the MRS broth medium was prepared, the medium was inoculated with 1% of each strain and cultured for 24 hours, absorbance was measured at 600 nm and a growth curve was drawn.

As a result, it could be confirmed that all Lactobacillus curvatus stains isolated from natural sourdough used maltose better than the reference strain (L. curvatus KCCM 40715). In particular, 104, 114C, 156SS and 183N exhibited superior maltose availability as compared to other strains, and absorbance values thereof were 1.498, 1.501, 1.523 and 1.528. The absorbance values were 0.253, 0.256, 0.278 and 0.283 higher than that of the reference strain.

Lactobacillus curvatus (L. curvatus) 104 strain found to exhibit high acid resistance and maltose availability in the testing was designated “Lactobacillus curvatus SPC-SNU 70-3” and then deposited under the accession number “KCTC 12778BP”.

Example 8 Selection of Superior Strain as Starter from Lactobacillus brevis (L. brevis) Isolated from Natural Sourdough—Confirmation of Acid Resistance at pH Levels and Maltose Availability

In the present example, acid resistance at pH levels and maltose availability of Lactobacillus brevis (L. brevis) strains were confirmed in order to select superior strain as starter from Lactobacillus brevis (L. brevis) isolated from natural sourdough.

(1) Confirmation of Acid Resistance at pH Levels with Respect to Lactobacillus brevis Isolated from Natural Sourdough.

Cell growth rates were primarily measured and compared at pH levels of 4 and 3.5 by microplate culture for Lactobacillus brevis strains isolated from natural sourdough. Of the strains, 111, 149 and T30 were selected as strains having the highest growth levels.

Then, MRS broth at pH levels of 4 and 3.5 was inoculated with 1% of each of the strains and cultured for 24 hours, absorbance was measured at 600 nm and a growth curve was drawn.

As a result, it could be confirmed that strains 111, 149 and T30 exhibited similar growth rates to the reference strain (L. brevis KCCM 11433), and of these, strains 111 and 149 exhibited higher growth rates than the reference strain. In particular, it could be confirmed that strain 149 having the highest growth rate was the most suitable as the starter (FIG. 7). FIG. 7 shows a confirmation result of acid resistance of Lactobacillus brevis isolated from natural sourdough.

(2) Confirmation of Maltose Availability of Lactobacillus brevis Isolated from Natural Sourdough

A 2% maltose-containing MRS broth medium having the composition shown in the following Table 1 was prepared, the medium was inoculated with 1% of each of strains 111, 149 and T30, and cultured for 24 hours, absorbance was measured at 600 nm and a growth curve was drawn.

TABLE 1 Composition Content Proteose peptone No.3 10.0 g Beef extract 10.0 g Yeast extract 5.0 g Maltose 20.0 g Polysorbate 80 1.0 g Ammonium citrate 2.0 g Sodium acetate 5.0 g Magnesium sulfate 0.1 g Manganese sulfate 0.05 g Dipotassium phosphate 2.0 g Distilled water 1 L

As a result, it could be confirmed that strains 149 and T30 were grown rapidly in maltose medium than the reference strain, and of these, strain 149 had the highest growth rate. The result showed that strain 149 was the most suitable as a starter (FIG. 8). FIG. 8 shows a confirmation result of maltose availability of Lactobacillus brevis isolated from natural sourdough.

Meanwhile, Lactobacillus brevis (L. brevis) strain 149 was designated “Lactobacillus brevis SPC-SNU 70-2” and then deposited under the accession number “KCTC 12777BP”.

Example 9 Selection of Superior Strain as Starter from Saccharomyces cerevisiae (S. cerevisiae) Isolated from Natural Sourdough—Confirmation of Acid Resistance at pH Levels

In the present example, superior strain as starter was selected from Saccharomyces cerevisiae (S. cerevisiae) isolated from natural sourdough.

First, Saccharomyces cerevisiae 01434 isolated from early natural sourdough (early sourdough) and Saccharomyces cerevisiae 01435 isolated from mature natural sourdough (mature sourdough) were tested. The test was conducted on a 96 microwell plate, 20 g/L maltose was added to a YP medium based on 250 μl of a total volume per well, and the bacteria was inoculated at an early OD₆₀₀ of 0.2. Acids used for resistance test were lactic acid and acetic acid, which are the most commonly used in sourdough, and were used at pH levels of 5.5, 5.0, 4.5, 4.0 and 3.5.

As a result, Saccharomyces cerevisiae 01434 was almost not grown in acetic acid at pH 3.5, was grown slowly at pH 4.0, and was grown without significant difference from pH 4.5 to 5.5. Unlike this, Saccharomyces cerevisiae 01434 was grown well regardless of acidity in lactic acid, in particular, was grown very well at pH 5.5. This result showed that Saccharomyces cerevisiae (S. cerevisiae) 01434 had lower resistance to acetic acid than resistance to lactic acid and its growth was inhibited in acetic acid at pH of 4.5 or less.

In addition, yeast isolated from early natural sourdough and yeast isolated from mature natural sourdough exhibited similar acid resistance and Saccharomyces cerevisiae 01435 isolated from mature natural sourdough exhibited slightly better acid resistance (FIG. 9). FIG. 9 shows a confirmation result of acid resistance of Saccharomyces cerevisiae 01435 isolated from natural sourdough.

Meanwhile, Saccharomyces cerevisiae (S. cerevisiae) 01435 was designated “Saccharomyces cerevisiae SPC-SNU 70-1” and then deposited under the accession number “KCTC 12776BP”.

Example 10 Application of Saccharomyces cerevisiae (S. cerevisiae) Isolated from Natural Sourdough to Breadmaking

In the present example, Saccharomyces cerevisiae 01435 (S. cerevisiae 01435), which was selected as the best strain as a starter for breadmaking, among Saccharomyces cerevisiae (S. cerevisiae) strains isolated from natural sourdough (Nuruk-containing Korean sourdough) and commercial yeast [ReusyaPro, (France), water content: 28%] were applied to bread making and characteristics thereof were compared.

(1) Bread Making

Ingredients constituting sponge dough shown in the following Table 2 were added to a mixer (SK101S MIXER®, Japan), kneaded in a second stage for 2 minutes, in a third stage for 1 minute, and further mixed until a final temperature of the kneaded substance reached 25° C. Then, the mixture was allowed to stand at room temperature for 30 minutes and primarily fermented at 6° C. in a fermenter for 16 hours to prepare sponge dough.

Then, ingredients constituting dough (strong flour, refined salt, refined sugar, whole milk powder, yeast and purified water) shown in the following Table 2 were put into a mixer (SK101S MIXER®, Japan) and kneaded in a first stage for 1 minute, the sponge dough was added thereto and the resulting mixture was further mixed in a second stage for 3 minutes and in a third stage for 2 minutes. Then, butter was added to the mixture, and the resulting mixture was kneaded in a second stage for 3 minutes and in a third stage for 3 minutes and further mixed until a final temperature of the kneaded substance reached 27° C. to prepare dough.

The dough was secondarily fermented in a fermenter at 27° C. and at relative humidity of 85% for 30 minutes, cut to a predetermined size, made round and aged in a fermenter at 27° C. and at relative humidity of 85% for 15 minutes. After aging, the dough was molded and put into a bread case. Then, the dough put into the bread case was fermented under the conditions of 37° C. and a relative humidity of 85% for 50 minutes to prepare a bread dough. The bread dough was baked in an oven at an upper heat of 170° C. and at a lower heat of 210° C. for 35 minutes. Then, the bread was cooled at room temperature until an inner temperature thereof reached 32° C.

Images of bread containing the Saccharomyces cerevisiae 01435 prepared by the process described above and commercial yeast are shown in FIG. 10 (FIG. 10). FIG. 10 shows images of control group (commercial yeast-applied bread) and isolated yeast Saccharomyces cerevisiae (S. cerevisiae) 01435-applied bread.

TABLE 2 Control group Isolated yeast (commercial (S. cerevisiae yeast- 01435)- Ingredients applied bread) applied bread Sponge Strong flour 70 70 dough Commercial yeast 0.7 — Isolated yeast — 1.6 Rimulsoft 0.3 0.3 Feed water 42 42 Dough Strong flour 30 30 Refined salt 1.8 1.8 Refined sugar 7 7 Whole milk powder 3 3 Butter 10 10 Commercial yeast 0.6 — Isolated yeast — 1.4 Feed water 23 22 (Unit: g)

(2) Measurement of Physical Properties of Bread

Physical properties were measured for the Saccharomyces cerevisiae 01435-applied bread and the commercial yeast-applied bread.

Measurement of pH was carried out using a pH meter in a 250 mL beaker which was filled with 100 mL of distilled water and 15 g of a sample and was then homogenized.

Total titratable acidity (TTA) was defined by an amount mL of a 0.1N NaOH solution which was consumed until pH reached 6.6 and 8.5 upon titration with the 0.1N NaOH solution.

Chromaticity was measured for a bread sample cut to a thickness of about 20 mm using a chromameter (CR-400, KONICA MINOLTA, Inc.). With regard to the measured chromaticity, lightness was represented by the value “L”, and chromaticity for red to green and for yellow to blue were represented by the value and the value “b”, respectively.

Measurement results of pH of bread, total titratable acidity and chromaticity are shown in the following Table 3.

TABLE 3 Control group Isolated yeast (commercial (S. cerevisiae yeast-applied 01435)-applied bread) bread pH  5.53  5.46 TTA (6.6/8.5) 2.27/4.87 2.43/4.91 Water content    41.67%    41.73% Secondary fermentation 55 minutes 55 minutes time Specific volume  4.92  4.98 Hunter lab L 85.11 84.11 color values a −2.06 −2.02 b 17.92 17.52

As a result, the Saccharomyces cerevisiae 01435-applied bread exhibited lower pH, higher water content and greater specific volume than the commercial yeast-applied bread.

(3) Confirmation of Gas Generation Capability of Dough

Gas generation capability was compared and confirmed between Saccharomyces cerevisiae 01435-applied dough and the commercial yeast-applied dough.

The gas generation capability was measured on 25 g of dough using a gas generation capability measurement device (fermometer) at 30° C. for 10 hours.

As a result of measurement, two types of dough exhibited overall similar gas generation capability. The Saccharomyces cerevisiae 01435-applied dough exhibited slightly high gas generation capability only in an early stage (FIG. 11). FIG. 11 is a graph showing gas generation capability of dough containing Saccharomyces cerevisiae isolated from natural sourdough.

(4) Measurement of Aging Level of Bread

Hardness and aging rate over time were compared between the Saccharomyces cerevisiae 01435-applied bread and the commercial yeast-applied bread.

The bread was stood at room temperature for 3 hours and was cut to a thickness of about 20 mm to prepare a sample, hardness of the sample was measured using a physical property measurement machine (Texture analyzer, Stable Micro Systems Ltd.) and hardness over time was compared to measure an aging rate. Hardness measurement results are shown in the following Table 4 and the aging rate is shown in FIG. 12.

TABLE 4 Sample Hardness On the 1^(st) day (after 19 hours) Control group (commercial yeast applied bread) 181.322 Isolated yeast (S. cerevisiae 01435)-applied bread 156.43 On the 3^(rd) day (after 63 hours) Control group (commercial yeast-applied bread) 260.353 Isolated yeast (S. cerevisiae 01435)-applied bread 222.876 On the 4^(th) day (after 87 hours) Control group (commercial yeast-applied bread) 281.774 Isolated yeast (S. cerevisiae 01435)-applied bread 241.639

As a result, it could be confirmed that the Saccharomyces cerevisiae 01435-applied bread had lower overall hardness than the commercial yeast-applied bread and thus excellent softness.

However, as a result of measurement of aging rate based on comparison in hardness over time, two breads had similar overall aging rate (FIG. 12). FIG. 12 shows a confirmation result of aging rate of bread containing Saccharomyces cerevisiae 01435 isolated from natural sourdough.

(5) Analysis of Aroma Ingredients of Bread

Aroma ingredients were analyzed using a GC/MS system to compare flavor and taste ingredients between the Saccharomyces cerevisiae 01435-applied bread and the commercial yeast-applied bread.

Analysis was conducted on 1 g of a sample and GC/MS analysis conditions are shown in the following Table 5. After GC/MS analysis, the overall quantitative values of alcohols, aldehydes, ketones, esters and acids were compared (FIG. 13), and relative proportions of 22 types of respective representative aroma ingredients are shown as percentages in the following Table 6.

TABLE 5 Analysis system Operation conditions GC/MS analysis GC Model name: Agilent 7890A Inlet temperature: 230° C. Column: DB-WAX (60 m × 250 μm × 0.25 μM) Carrier gas: helium Flow rate: 1 ml/min Oven temperature program: from 40° C. (5 min) → 8° C./min → 230° C. (10 min) MS detector: Agilent 5975C MSD (EI mode) SPME analysis Fiber: DVB/Carboxen/PSME (Supelco Co.) Sample equilibration time incubation temp. 85° C. incubation time 30 min

TABLE 6 Content proportion (%) of Content proportion (%) of aroma ingredients contained aroma ingredients of isolated in control group (commercial yeast (S. cerevisiae 01435)- Aroma ingredients yeast-applied bread) applied bread Alcohol Ethyl alcohol 59.89 90.11 43.46 86.37 1-Propanol 0.55 0.47 2-Methyl-1-propanol 3.55 2.46 Isoamyl alcohol 15.89 15.68 1-Hexanol 0.71 1.24 2-Phenyl ethyl alcohol 9.52 23.04 Aldehyde Hexanal 0.33 2.58 0.61 3.78 Nonanal 0.56 0.57 Furfural 0.16 0.36 Benzaldehyde 1.53 2.25 Ketone 2-Heptanone 0.50 2.45 0.87 4.71 Acetoin 1.38 3.10 2-Nonanone 0.56 0.75 Ester Ethyl hexanoate 1.00 4.34 1.03 4.29 Ethyl octanoate 2.69 2.33 Ethyl decanoate 0.31 0.35 Isoamyl lactate 0.34 0.57 Acid Octanoic acid 0.22 0.32 2.33 0.33 Acetic acid 0.10 0.35 Hexanoic acid 0.00 0.57 Others Alpha-limonene 0.20 0.20 0.52 0.21 Total 100 100 100 100

As a result of comparison of overall quantitative values of volatile aroma ingredients (alcohols, aldehydes, ketones, esters and acids), it could be confirmed that the Saccharomyces cerevisiae 01435-applied bread contained great amounts of ketones. Ketones are aroma ingredients having soft and mild fragrance and the isolated yeast-applied bread according to the present invention offered soft and mild flavor and taste. In particular, the isolated yeast-applied bread had a high content of acetoin among ketone having soft buttery taste. In addition, the Saccharomyces cerevisiae 01435-applied bread had a low content of ethyl alcohol having fresh fragrance, as compared to the control group.

Meanwhile, it could be confirmed that the commercial yeast-applied bread contained great amounts of alcohols and esters. Alcohols and esters are aroma ingredients having light and strong fragrance and the commercial yeast-applied bread had worse flavor and taste spectrum than the isolated yeast according to the present invention (FIG. 13). FIG. 13 shows a comparison result of quantitative values of aroma ingredients of bread containing Saccharomyces cerevisiae 01435 isolated from natural sourdough.

Example 11 Application of Lactobacillus curvatus (L. curvatus) Isolated from Natural Sourdough to Breadmaking

In the present example, a Lactobacillus curvatus (L. curvatus) 104 strain isolated from natural sourdough (Nuruk-containing Korean sourdough) and a Lactobacillus curvatus KCCM 40715 strain as a reference strain were applied to bread making and characteristics thereof were compared.

(1) Production and Analysis of Dough Fermented with Lactic Acid Bacteria

100 g of a strong flour was mixed with 2×10¹⁰ cfu/g of lactic acid bacteria and 100 g of heating/cooling water, the mixture was fermented at 30° C. in a fermenter to prepare dough fermented with lactic acid bacteria, the dough was cooled when pH thereof reached 4.2±0.2, and pH, TTA and the number of bacteria of lactic acid bacteria-fermented dough were measured.

Meanwhile, the lactic acid bacteria used in the present example were obtained by culturing in MRS broth at a temperature of 30° C. for 22±2 hours, centrifuging and washing with physiological saline, and the dough was first inoculated with 1×10⁸ cfu of the lactic acid bacteria per 1 g of the dough.

Test results are shown in the following Table 7.

TABLE 7 TTA (based on 15 g, mL) Number of bacteria pH pH 6.6 pH 8.5 (cfu/g) Isolated strain (L. curvatus 104)-applied dough 4.36 6.89 11.09 1.3 × 10⁹ Reference strain (L. curvatus KCCM 40715)-applied dough 4.38 8.48 11.99 1.4 × 10⁹

As a result of measurement, fermentation times of isolated strain (L. curvatus 104)-applied dough and the reference strain (L. curvatus KCCM 40715)-applied dough were about 3 hours and the isolated strain-applied dough contained a similar number of bacteria to the well-known strain-applied dough, as can be seen from Table 7.

(2) Bread Making

Ingredients constituting sponge dough as shown in the following Table 8 were put into a mixer (SK101S MIXER®, Japan), kneaded in a second stage for 2 minutes and in a third stage for 1 minute and then further mixed until the final temperature of the kneaded substance reached 25° C. Then, the mixture was allowed to stand at room temperature for 30 minutes and primarily fermented at 6° C. in a fermenter for 16 hours to prepare sponge dough.

Then, ingredients constituting dough (strong flour, refined salt, refined sugar, whole milk powder, yeast, purified water and lactic acid bacteria-fermented dough) as shown in the following Table 8 were put into a mixer (SK101S MIXER®, Japan) and kneaded in a first stage for 1 minute, the sponge dough was added thereto and the resulting mixture was further mixed in a second stage for 3 minutes and in a third stage for 2 minutes. Then, butter was added to the mixture, and the resulting mixture was kneaded in the second stage for 3 minutes and in the third stage for 3 minutes until the final temperature of the kneaded substance reached 27° C. to prepare dough.

The dough was secondarily fermented in a fermenter at 27° C. and at relative humidity of 85% for 30 minutes, cut to a predetermined size, made round and aged in a fermenter at 27° C. and at relative humidity of 85% for 15 minutes. After aging, the dough was molded and put into a bread case. Then, the dough put into the bread case was fermented under the conditions of 37° C. and a relative humidity of 85% for 50 minutes to prepare a bread dough. The bread dough was baked in an oven at an upper heat of 170° C. and at a lower heat of 210° C. for 35 minutes. Then, the bread was cooled at room temperature until an inner temperature thereof reached 32° C.

Images of a produced control group (only commercial yeast-applied bread), reference strain (L. curvatus KCCM40715)-applied bread and isolated strain (L. curvatus 104)-applied bread are shown in FIG. 14 (FIG. 14). FIG. 14 shows images of the control group (only commercial yeast-applied bread), the reference strain (L. curvatus KCCM40715)-applied bread and the isolated strain (L. curvatus 104)-applied bread.

TABLE 8 Control Isolated group Reference strain (L. (commercial strain (L. curvatus yeast- curvatus 104)- applied KCCM40715)- applied Ingredients bread) applied bread bread Sponge Strong flour 70 70 70 dough Commercial yeast 0.7 0.7 0.7 Rimulsoft 0.3 0.3 0.3 Purified water 42 42 42 Dough Strong flour 30 20 20 Refined salt 1.8 1.8 1.8 Refined sugar 7 7 7 Whole milk powder 3 3 3 Butter 10 10 10 Commercial yeast 0.6 0.6 0.6 Purified water 23 13 13 Lactic acid bacteria- — 20 20 fermented dough (Unit: g)

(3) Measurement of Physical Properties of Bread

Physical properties (pH, total titratable acidity and chromaticity) of the control group (only commercial yeast-applied bread), the reference strain (L. curvatus KCCM40715)-applied bread and the isolated strain (L. curvatus 104)-applied bread were measured. Measurement of pH, total titratable acidity and chromaticity was carried out using the same method as in Example 10 and results are shown in the following Table 9.

TABLE 9 Control group Reference strain Isolated strain (commercial (L. curvatus (L.curvatus yeast KCCM40715)- 104)- applied bread) applied bread applied bread pH pH 5.53 pH 5.41 pH 5.39 TTA (6.6/8.5) 2.27/4.87 2.51/5.22 2.55/5.39 Water content    41.67%    41.70%    41.71% Secondary fermentation 55 minutes 52 minutes 52 minutes time Specific volume  4.92  4.98  5.01 Hunter lab L 85.11 84.11 85.9 color values a −2.06 −2.66 −2.58 b 17.92 17.03 16.55

As a result, it could be confirmed that the isolated strain-applied bread had lower pH and greater specific volume than the reference strain-applied bread.

(4) Confirmation of Gas Generation Capability of Dough

Gas generation capability was compared between the control group (only commercial yeast-applied dough), reference strain (L. curvatus KCCM40715)-applied dough and isolated strain (L. curvatus 104)-applied dough. The gas generation capability was measured on 25 g of dough using a gas generation capability measurement device (fermometer) at 30° C. for 10 hours.

As a result of measurement, it could be confirmed that the lactic acid bacteria strain (reference strain, isolated strain)-applied bread dough exhibited better gas generation capacity than the control group (only commercial yeast-applied bread dough. It could be confirmed that there was almost no difference between the isolated strain and the reference strain (FIG. 15). FIG. shows a confirmation result of gas generation capability of dough containing Lactobacillus curvatus 104 (L. curvatus 104) isolated from natural sourdough.

(5) Measurement of Aging Level of Bread

Hardness and aging rate over time were compared between the control group (only commercial yeast-applied bread), reference strain (L. curvatus KCCM40715)-applied bread and isolated strain (L. curvatus 104)-applied bread.

Hardness and aging rate over time were measured in the same manner as in Example 10, hardness values are shown in the following Table 10 and results of aging rate over time are shown in FIG. 16.

TABLE 10 Sample Hardness On the 1^(st) day (after 19 hours) Control group (only commercial yeast-applied 181.322 bread) Reference strain (L. curvatus KCCM40715)- 172.441 applied bread Isolated strain (L. curvatus 104)-applied bread 162.7 On the 3^(rd) day (after 63 hours) Control group (only commercial yeast-applied 260.353 bread) Reference strain (L. curvatus KCCM40715)- 252.077 applied bread Isolated strain (L. curvatus 104)-applied bread 234.832 On the 4^(th) day (after 87 hours) Control group (only commercial yeast-applied 281.774 bread) Reference strain (L. curvatus KCCM40715)- 274.799 applied bread Isolated strain (L. curvatus 104)-applied bread 254.512

As a result, it could be confirmed that the lactic acid bacteria strain (reference strain and isolated strain)-applied breads had low hardness and were thus soft. In particular, it could be confirmed that the isolated strain (L. curvatus 104)-applied bread had the lowest hardness and was thus the softest. In addition, it could be confirmed that results of aging rate over time were similar to those of hardness (FIG. 16). FIG. 16 shows a confirmation result of aging rate of bread containing Lactobacillus curvatus 104 (L. curvatus 104) isolated from natural sourdough.

(6) Analysis of Aroma Ingredients of Bread

Aroma ingredients were analyzed using a GC/MS system to compare expression of flavor and taste ingredients between the control group (only commercial yeast-applied bread), the reference strain (L. curvatus KCCM40715)-applied bread, the isolated strain (L. curvatus 104)-containing bread.

Analysis was conducted on 1 g of a sample and GC/MS analysis conditions were the same as in Table 5 given above. After GC/MS analysis, the overall quantitative values of alcohols, aldehydes, ketones, esters and acids were compared (FIG. 17), and relative proportions of 22 types of respective representative aroma ingredients are shown as percentages in the following Table 11.

TABLE 11 Content Content Content proportion proportion proportion (%) of aroma (%) of aroma (%) of aroma ingredients ingredients ingredients contained in contained in contained in control group isolated reference strain (only commercial strain (L. (L. curvatus yeast-applied curvatus 104)- KCCM 40715)- Aroma ingredients bread) applied bread applied bread Alcohol Ethyl alcohol 55.45 85.69 42.22 83.37 44.03 84.84 1-Propanol 0.73 0.71 0.00 2-Methyl-1-propanol 4.31 4.12 3.78 Isoamyl alcohol 10.49 22.39 21.47 1-Hexanol 1.01 1.74 1.29 2-Phenyl ethyl 13.71 12.20 14.26 alcohol Aldehyde Hexanal 0.47 3.80 0.62 4.57 0.52 3.99 Nonanal 0.85 0.91 0.91 Furfural 0.28 0.52 0.61 Benzaldehyde 2.20 2.52 1.95 Ketone 2-Heptanone 0.42 2.84 0.86 4.17 0.70 3.81 Acetoin 1.59 2.26 2.05 2-Nonanone 0.82 1.04 1.05 Ester Ethyl hexanoate 1.70 6.36 1.62 5.88 1.68 5.85 Ethyl octanoate 4.04 3.41 3.85 Ethyl decanoate 0.62 0.27 0.33 Isoamyl lactate 0.00 0.58 0.00 Acid Octanoic acid 0.51 0.87 1.34 1.57 0.40 1.51 Acetic acid 0.36 0.23 0.19 Hexanoic acid 0.00 0.00 0.92 Others Alpha-limonene 0.44 0.44 0.44 0.44 0.00 0.00 Total 100 100 100 100 100 100

As a result of comparison of overall quantitative values of volatile aroma ingredients (alcohols, aldehydes, ketones, esters and acids), it could be confirmed that the (L. curvatus 104)-applied bread contained great amounts of ketones. Ketones are aroma ingredients having soft and mild fragrance and it could be confirmed that the bread containing the isolated strain according to the present invention offered soft and mild flavor and taste.

Meanwhile, it could be confirmed that the lactic acid bacteria (isolated strain, reference strain)-applied breads contained great amounts of acids.

On the other hand, it could be confirmed that only commercial yeast-applied bread contained great amounts of alcohols and esters. Alcohols and esters are aroma ingredients having light and strong fragrance and it could be confirmed that the commercial yeast-applied bread had worse flavor and taste spectrum.

FIG. 17 shows results of comparison of quantitative values of aroma ingredients of bread containing Lactobacillus curvatus (L. curvatus 104) isolated from natural sourdough.

Meanwhile, as a result of analysis of representative aroma ingredients, as can be seen from Table 11, the lactic acid bacteria (isolated strain, reference strain)-applied breads had a low content of ethyl alcohol having fresh fragrance, as compared to the control group (only commercial yeast-applied bread). In addition, nonanal having citrus and fatty flavors (pleasant flavors), and furfural having caramel, candy and almond flavors were found to be present in higher amounts in the lactic acid bacteria (isolated strain, Reference strain)-applied bread. In particular, benzaldehyde having sweet flavor and taste like candy flavor and acetoin of ketone having soft buttery taste were found to be present in higher amounts in the isolated strain (L. curvatus 104). In addition, it could be confirmed that the reference strain (L. curvatus kccm 40715)-applied bread contained neither 1-propanol having sweet fragrance nor alpha-limonene having orange fragrance.

Example 12 Application of Lactobacillus brevis (L. brevis) Isolated from Natural Sourdough to Breadmaking

In the present example, a Lactobacillus brevis (L. brevis) 149 strain isolated from natural sourdough (Nuruk-containing Korean sourdough) and a reference strain (L. brevis KACC 11433) were applied to bread making and characteristics thereof were compared.

(1) Production and Analysis of Dough Fermented with Lactic Acid Bacteria

100 g of a strong flour was mixed with 2×10¹⁰ cfu/g of lactic acid bacteria and 100 g of heating/cooling water, the mixture was fermented at 30° C. in a fermenter to prepare dough fermented with lactic acid bacteria, the dough was cooled when pH thereof reached 4.2±0.2, and pH, TTA and the number of bacteria of lactic acid bacteria-fermented dough were measured.

Meanwhile, the lactic acid bacteria strains were bacteria obtained by culturing in MRS broth at a temperature of 30° C. temperature for 22±2 hours, centrifuging and washing with physiological saline and the dough was first inoculated with 1×10⁸ cfu of the lactic acid bacteria per 1 g of the dough.

Test results are shown in the following Table 12.

TABLE 12 TTA (based on 15 g, mL) Number of bacteria pH pH 6.6 pH 8.5 (cfu/g) Isolated strain (L. brevis 149)-applied dough 4.42 7.25 9.75 1.1 × 10⁹ Reference strain (L. brevis KACC 11433)-applied dough 4.42 5.34 7.47 6.1 × 10⁸

As a result of measurement, fermentation time of isolated strain (L. brevis 149)-applied dough was 6 hours and reference strain (L. brevis KACC 11433)-applied dough was 8 hours.

In addition, as can be seen from Table 12 given above, the isolated strain-applied dough had about a 1.8-fold higher number of bacteria than the reference strain-applied dough.

(2) Bread Making

Ingredients constituting sponge dough as shown in the following Table 13 were put into a mixer (SK101S MIXER®, Japan), kneaded in a second stage for 2 minutes and in a third stage for 1 minute and then further mixed until the final temperature of the kneaded substance reached 25° C. Then, the mixture was allowed to stand at room temperature for 30 minutes and primarily fermented at 6° C. in a fermenter for 16 hours to prepare sponge dough.

Then, ingredients constituting dough (strong flour, refined salt, refined sugar, whole milk powder, yeast, purified water and lactic acid bacteria-fermented dough) as shown in the following Table 13 were put into a mixer (SK101S MIXER®, Japan), kneaded in a first stage for 1 minute, the sponge dough was added thereto and the resulting mixture was further mixed in a second stage for 3 minutes and in a third stage for 2 minutes. Then, butter was added to the mixture, and the resulting mixture was kneaded in the second stage for 3 minutes and in the third stage for 3 minutes until the final temperature of the kneaded substance reached 27° C. to prepare dough.

The dough was secondarily fermented in a fermenter at 27° C. and at relative humidity of 85% for 30 minutes, cut to a predetermined size, made round and aged in a fermenter at 27° C. and at relative humidity of 85% for 15 minutes. After aging, the dough was molded and put into a bread case. Then, the dough put into the bread case was fermented under the conditions of 37° C. and a relative humidity of 85% for 50 minutes to prepare a bread dough. The bread dough was baked in an oven at an upper heat of 170° C. and at a lower heat of 210° C. for 35 minutes. Then, the bread was cooled at room temperature until an inner temperature thereof reached 32° C.

Images of a produced control group (only commercial yeast-applied bread), reference strain (L. brevis KACC 11433)-applied bread and isolated strain (L. brevis 149)-applied bread are shown in FIG. 18 (FIG. 18). FIG. 18 shows images of the control group (only commercial yeast-applied bread), the reference strain (L. brevis KACC 11433)-applied bread and the isolated strain (L. brevis 149)-applied bread.

TABLE 13 Control Reference group strain Isolated (only (L. brevis strain commercial KACC (L. brevis yeast- 11433)- 149)- applied applied applied Ingredients bread) bread bread Sponge Strong flour 70 70 70 dough commercial yeast 0.7 0.7 0.7 Rimulsoft 0.3 0.3 0.3 Purified water 42 42 42 Dough Strong flour 30 20 20 Refined salt 1.8 1.8 1.8 Refined sugar 7 7 7 Whole milk powder 3 3 3 Butter 10 10 10 Commercial yeast 0.6 0.6 0.6 Purified water 23 13 13 Lactic acid bacteria- — 20 20 fermented dough (Unit: g)

(3) Measurement of Physical Properties of Bread

Physical properties (pH, total titratable acidity and chromaticity) of the control group (only commercial yeast-applied bread), the reference strain (L. brevis KACC 11433)-applied bread and the isolated strain (L. brevis 149)-applied bread were measured. Measurement of pH, total titratable acidity and chromaticity was carried out using the same method as in Example 10 and results are shown in the following Table 14.

TABLE 14 Control group Reference Isolated (only strain (L. strain commercial brevis KACC (L. brevis yeast-applied 11433)- 149)-applied bread) applied bread bread pH 5.53 5.25 5.29 TTA (6.6/8.5) 2.27/4.87 3.65/6.86 3.54/6.61 Water content    41.67%    42.45%    42.39% Secondary 55 minutes 56 minutes 56 minutes fermentation time Specific volume  4.92  5.12  5.15 Hunter lab L 85.11 84.19 85.92 color values a −2.06 −2.12 −2.35 b 17.92 17.09 16.55

As a result, it could be confirmed that the isolated strain-applied bread had higher pH and greater specific volume than the reference strain-applied bread.

(4) Confirmation of Gas Generation Capability of Dough

Gas generation capability was compared and confirmed between the control group (only commercial yeast-applied dough), the reference strain (L. brevis KACC 11433)-applied dough, and isolated strain (L. brevis 149)-applied dough. The gas generation capability was measured on 25 g of dough using a gas generation capability measurement device (fermometer) at 30° C. for 10 hours.

As a result of measurement, the lactic acid bacteria strain (reference strain, isolated strain)-applied dough exhibited better gas generation capacity than the control group (only commercial yeast-applied dough). It could be confirmed that there was almost no difference between the isolated strain and the reference strain (FIG. 19). FIG. 19 shows a confirmation result of gas generation capability of dough containing Lactobacillus brevis (L. brevis 149) isolated from natural sourdough.

(5) Measurement of Aging Level of Bread

Hardness and aging rate over time were compared between the control group (only commercial yeast-applied bread), the reference strain (L. brevis KACC 11433)-applied bread and the isolated strain (L. brevis 149)-applied bread.

Hardness and aging rate over time were measured in the same manner as in Example 10, hardness values are shown in the following Table 15 and results of aging rate over time are shown in FIG. 20.

TABLE 15 Sample Hardness On the 1^(st) day (after 19 hours) Control group (only commercial yeast- 181.322 applied bread) Reference strain (L. brevis KACC 11433)- 159.055 applied bread Isolated strain (L. brevis 149)-applied 155.287 bread On the 3^(rd) day (after 63 hours) Control group (only commercial yeast- 283.548 applied bread) Reference strain (L. brevis KACC 11433)- 267.551 applied bread Isolated strain (L. brevis 149)-applied 243.507 bread On the 4^(th) day (after 87 hours) Control group (only commercial yeast- 288.54 applied bread) Reference strain (L. brevis KACC 11433)- 275.595 applied bread Isolated strain (L. brevis 149)-applied 253.402 bread

As a result, it was confirmed that the lactic acid bacteria strain (reference strain, isolated strain)-applied breads had a low hardness and were thus soft. In particular, the isolated strain (L. brevis 149)-applied bread had the lowest hardness.

Meanwhile, as a result of analysis of aging time over time, it was confirmed that lactic acid bacteria-applied breads had lower aging rates than the control group and in particular, the isolated strain-applied bread had the lowest aging rate (FIG. 20). FIG. 20 shows a confirmation result of aging rate of bread containing Lactobacillus brevis (L. brevis 149) isolated from natural sourdough.

(6) Analysis of Aroma Ingredients of Bread

Aroma ingredients were analyzed using a GC/MS system to compare flavor and taste ingredients between the control group (only commercial yeast-applied bread), the reference strain (L. brevis KACC 11433)-applied bread and the isolated strain (L. brevis 149)-applied bread.

Analysis was conducted on 1 g of a sample and GC/MS analysis conditions were the same as in Table 5 given above. After GC/MS analysis, the overall quantitative values of alcohols, aldehydes, ketones, esters and acids were compared (FIG. 21), and relative proportions of 22 types of respective representative aroma ingredients are shown as percentages in the following Table 16.

TABLE 16 Content Content Content proportion proportion proportion (%) of aroma (%) of aroma (%) of aroma ingredients ingredients ingredients contained in contained in contained in reference strain control group (only isolated strain (L. (L. brevis KACC commercial yeast- brevis 149)-applied 11433)-applied Aroma ingredients applied bread) bread bread Alcohol Ethyl alcohol 59.89 90.11 51.23 85.90 51.77 87.24 1-Propanol 0.55 0.56 0.54 2-Methyl-1-propanol 3.55 3.39 3.56 Isoamyl alcohol 15.89 16.91 16.97 1-Hexanol 0.71 0.78 0.78 2-Phenyl ethyl 9.52 13.03 13.62 alcohol Aldehyde Hexanal 0.33 2.58 0.50 2.76 0.43 2.71 Nonanal 0.56 0.83 0.65 Furfural 0.16 0.41 0.32 Benzaldehyde 1.53 1.01 1.31 Ketone 2-Heptanone 0.50 2.45 0.41 2.73 0.35 2.63 Acetorin 1.38 1.57 1.65 2-Nonanone 0.56 0.75 0.63 Ester Ethyl hexanoate 1.00 4.34 1.53 6.72 1.50 6.06 Ethyl octanoate 2.69 4.12 4.07 Ethyl decanoate 0.31 0.50 0.49 Isoamyl lactate 0.34 0.58 0.00 Acid Octanoic acid 0.22 0.32 0.46 1.70 0.30 1.18 Acetic acid 0.10 0.45 0.24 Hexanoic acid 0.00 0.79 0.64 Others Alpha-limonene 0.20 0.20 0.19 0.19 0.19 0.19 Total 100 100.00 100 100 100 100

As a result of comparison of overall quantitative values of volatile aroma ingredients (alcohols, aldehydes, ketones, esters and acids), it could be confirmed that the lactic acid bacteria (isolated strain and reference strain)-applied breads contained great amounts of ketones, aldehydes and esters. Ketones, aldehydes and esters are aroma ingredients having soft and mild fragrance and it could be confirmed that the isolated strain (L. brevis 149)-applied bread according to the present invention offered soft and mild flavor and taste.

In addition, it could be confirmed that the lactic acid bacteria (isolated strain, reference strain)-applied breads contained great amounts of acids.

On the other hand, it could be confirmed that only commercial yeast-applied bread contained great amounts of alcohols. Alcohols are aroma ingredients having light and strong fragrance and it could be confirmed that the commercial yeast-applied bread had worse flavor and taste.

FIG. 21 shows results of comparison of quantitative values of aroma ingredients of bread containing Lactobacillus brevis (L. brevis 149) isolated from natural sourdough.

Meanwhile, as a result of analysis of representative aroma ingredients, as can be seen from Table 16 given above, the lactic acid bacteria (isolated strain, reference strain)-applied breads had a low content of ethyl alcohol having fresh fragrance, as compared to the control group (only commercial yeast-applied bread).

In addition, isoamyl alcohol having banana or European pear-like sweet flavor, 2-phenyl ethyl alcohol having corn-like sweet flavor, and acetoin of ketone having soft buttery taste were found to be present in higher amounts of lactic acid bacteria-containing breads. In addition, nonanal having citrus and fatty flavors (pleasant flavors), furfural having caramel, candy and almond flavors and esters having sweet flavor were found to be present in higher amounts in isolated strain (L. brevis 149)-applied breads.

Example 13 Application of Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 Isolated from Natural Sourdough to Breadmaking

In the present example, a Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 strain isolated from natural sourdough (Nuruk-containing Korean sourdough) and a reference strain (L. sanfranciscensis KACC 12431) were applied to bread making and characteristics thereof were compared.

(1) Production and Analysis of Dough Fermented with Lactic Acid Bacteria

100 g of a strong flour was mixed with 2×10¹⁰ cfu/g of lactic acid bacteria and 100 g of heating/cooling water, the mixture was fermented at 30° C. in a fermenter to prepare dough fermented with lactic acid bacteria, the dough was cooled when pH thereof reached 4.2±0.2, and pH, TTA and the number of bacteria of lactic acid bacteria-fermented dough were measured. At this time, measurement of pH and TTA was carried out using the method described in Example 10 above.

Meanwhile, the lactic acid bacteria strains were obtained by culturing in MRS broth at a temperature of 30° C. for 22±2 hours, centrifuging and washing with physiological saline, and the dough was first inoculated with 1×10⁸ cfu of the lactic acid bacteria per 1 g of the dough.

Test results are shown in the following Table 17.

TABLE 1 TTA (based on 15 g, mL) Number of bacteria pH pH 6.6 pH 8.5 (cfu/g) Isolated strain (L. sanfranciscensis 142)-applied dough 4.41 6.41 9.17 1.4 Reference strain (L. sanfranciscensis KACC 12431)-applied dough 4.47 6.53 9.26 1.2

As a result of measurement, the fermentation time of isolated strain (L. sanfranciscensis 142)-applied dough was 6 hours and the reference strain (L. sanfranciscensis KACC 12431)-applied dough were 8 hours and the isolated strain-applied dough contained 2 hours faster than the reference strain-applied dough.

In addition, as can be seen from Table 17 given above, the isolated strain-applied dough had more bacteria than the reference strain-applied dough.

(2) Bread Making

Ingredients constituting sponge dough as shown in the following Table 18 were put into a mixer (SK101S MIXER®, Japan), kneaded in a second stage for 2 minutes and in a third stage for 1 minute and then further mixed until the final temperature of the kneaded substance reached 25° C. Then, the mixture was allowed to stand at room temperature for 30 minutes and primarily fermented at 6° C. in a fermenter for 16 hours to prepare sponge dough.

Then, ingredients constituting dough (strong flour, refined salt, refined sugar, whole milk powder, yeast, purified water and lactic acid bacteria-fermented dough) as shown in the following Table 18 were put into a mixer (SK101S MIXER®, Japan), kneaded in a first stage for 1 minute, the sponge dough was added thereto and the resulting mixture was further mixed in a second stage for 3 minutes and in a third stage for 2 minutes. Then, butter was added to the mixture, and the resulting mixture was kneaded in the second stage for 3 minutes and in the third stage for 3 minutes until the final temperature of the kneaded substance reached 27° C. to prepare dough.

The dough was secondarily fermented in a fermenter at 27° C. and at relative humidity of 85 to 90% for 30 minutes, cut to a predetermined size, made round and aged in a fermenter at 27° C. and at relative humidity of 85 to 90% for 15 minutes. After aging, the dough was molded and put into a bread case. Then, the dough put into the bread case was fermented under the conditions of 37° C. and a relative humidity of 85 to 90% for 50 to 60 minutes to prepare a bread dough. The bread dough was baked in an oven at an upper heat of 170° C. and at a lower heat of 210° C. for 35 minutes. Then, the bread was cooled at room temperature until an inner temperature thereof reached 32° C.

Images of a produced control group (only commercial yeast-applied bread), the reference strain (L. sanfranciscensis KACC 12431)-applied bread, and isolated strain (L. sanfranciscensis 142)-applied bread are shown in FIG. 22 (FIG. 22). FIG. 22 shows images of the control group (only commercial yeast-applied bread), the reference strain (L. sanfranciscensis KACC 12431)-applied bread and the isolated strain (L. sanfranciscensis 142)-applied bread.

TABLE 18 Control group Reference Isolated (only strain strain commercial (L. (L. yeast- sanfranciscensis sanfranciscensis Composition applied KACC 12431)- 142)-applied ingredients bread) applied bread bread Sponge Strong flour 70 70 70 dough Commercial 0.7 0.7 0.7 yeast Rimulsoft 0.3 0.3 0.3 Purified water 42 42 42 Dough Strong flour 30 20 20 Refined salt 1.8 1.8 1.8 Refined sugar 7 7 7 Whole milk 3 3 3 powder Butter 10 10 10 Commercial 0.6 0.6 0.6 yeast Purified water 23 13 13 Lactic acid — 20 20 bacteria- fermented dough

(3) Measurement of Physical Properties of Bread

Physical properties (pH, total titratable acidity and chromaticity) of the control group (only commercial yeast-applied bread), (L. sanfranciscensis KACC 12431)-applied bread and the isolated strain (L. sanfranciscensis 142)-applied bread were measured.

Measurement of pH, total titratable acidity and chromaticity was carried out using the same method as in Example 10 and results are shown in the following Table 19.

TABLE 19 Control Reference strain group (only (L. sanfranciscensis Isolated strain commercial yeast- KACC 12431)- (L. sanfranciscensis applied bread) applied bread 142)-applied bread pH  5.53  5.29  5.19 TTA (6.6/8.5) 2.27/4.87 3.55/6.15 4.56/7.57 Water content    41.67%    41.25%    41.28% Secondary fermentation 55 minutes 54 minutes 53 minutes time Specific volume  4.92  5.01  5.03 Hunter lab L 85.11 84.32 85.01 color values a −2.06 −2.19 −2.21 b 17.92 17.11 16.88

As a result, it could be confirmed that the isolated strain-applied bread had lower pH and greater specific volume than the reference strain-applied bread.

(4) Confirmation of Gas Generation Capability of Dough

Gas generation capability was compared between the control group (only commercial yeast-applied dough), reference strain (L. sanfranciscensis KACC 12431)-applied dough and isolated strain (L. sanfranciscensis 142)-applied dough. The gas generation capability was measured on 25 g of dough using a gas generation capability measurement device (fermometer) at 30° C. for 10 hours.

As a result of measurement, it could be confirmed that the lactic acid bacteria strain (reference strain, isolated strain)-applied dough exhibited better gas generation capacity than the control group (only commercial yeast-applied dough). It could be confirmed that there was almost no difference between the isolated strain and the reference strain (FIG. 23). FIG. 23 shows a confirmation result of gas generation capability of dough containing Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 isolated from natural sourdough.

(5) Measurement of Aging Level of Bread

Hardness and aging rate over time were compared between the control group (only commercial yeast-applied bread), the reference strain (L. sanfranciscensis KACC 12431)-applied bread, and isolated strain (L. sanfranciscensis 142)-applied bread.

Hardness and aging rate over time were measured in the same manner as in Example 10, hardness values are shown in the following Table 20 and results of aging rate over time are shown in FIG. 24.

TABLE 20 Sample Hardness On the 1^(st) day (after 19 hours) Control group (only commercial yeast- 181.322 applied bread) Reference strain (L. sanfranciscensis 166.204 KACC 12431)-applied bread Isolated strain (L. sanfranciscensis 142)- 161.019 applied bread On the 3^(rd) day (after 63 hours) Control group (only commercial yeast- 260.548 applied bread) Reference strain (L. sanfranciscensis 252.32 KACC 12431)-applied bread isolated strain (L. sanfranciscensis 142)- 251.052 applied bread On the 4^(th) day (after 87 hours) Control group (only commercial yeast- 288.54 applied bread) Reference strain (L. sanfranciscensis 277.873 KACC 12431)-applied bread isolated strain (L. sanfranciscensis 142)- 275.314 applied bread

As a result, it could be confirmed that the lactic acid bacteria strain (reference strain and isolated strain)-applied breads had low hardness and were thus soft. In particular, it could be confirmed that the isolated strain (L. sanfranciscensis 142)-applied bread had the lowest hardness and was thus the softest. In addition, as a result of analysis results of aging rate over time, it could be confirmed that that the lactic acid bacteria strain-applied breads had lower aging rate than the control group (FIG. 24). FIG. 24 shows a confirmation result of aging rate of bread containing Lactobacillus sanfranciscensis (L. sanfranciscensis 142) isolated from natural sourdough.

(6) Analysis of Aroma Ingredients of Bread

Aroma ingredients were analyzed using a GC/MS system to compare flavor and taste ingredients between the control group (only commercial yeast-applied bread), the reference strain (L. sanfranciscensis KACC 12431)-applied bread, and isolated strain (L. sanfranciscensis 142)-applied bread.

Analysis was conducted on 1 g of a sample and GC/MS analysis conditions were the same as in Table 5 given above. After GC/MS analysis, the overall quantitative values of alcohols, aldehydes, ketones, esters and acids were compared (FIG. 25), and relative proportions of 22 types of respective representative aroma ingredients are shown as percentages in the following Table 21.

TABLE 21 Content Content Content proportion (%) proportion (%) proportion (%) of aroma of aroma of aroma ingredients ingredients ingredients contained in contained in contained in control group isolated strain reference strain (only (L. sanfran- (L. Sanfran- commercial ciscensis ciscensis yeast-applied 142)-applied KACC 12431)- Aroma ingredients bread) bread applied bread Alcohol Ethyl alcohol 59.89 15.89 52.03 86.60 51.18 86.28 1-Propanol 0.55 0.51 0.52 2-Methyl-1- 3.55 3.66 3.91 propanol Isoamyl alcohol 90.11 18.26 19.08 1-Hexanol 0.71 0.89 1.08 2-Phenyl ethyl 9.52 11.25 10.52 alcohol Aldehyde Hexanal 0.33 2.58 0.47 2.68 0.45 2.73 Nonanal 0.56 0.54 0.59 Furfural 0.16 0.38 0.51 Benzaldehyde 1.53 1.29 1.18 Ketone 2-Heptanone 0.50 2.45 0.36 2.40 0.34 2.42 Acetoin 1.38 1.43 1.48 2-Nonanone 0.56 0.60 0.59 Ester Ethyl hexanoate 1.00 4.34 1.68 7.42 1.83 7.36 Ethyl octanoate 2.69 4.71 4.73 Ethyl decanoate 0.31 0.48 0.47 Isoamyl lactate 0.34 0.55 0.32 Acid Octanoic acid 0.22 0.32 0.33 0.59 0.00 0.95 Acetic acid 0.10 0.26 0.00 Hexanoic acid 0.00 0.00 0.95 Others Alpha-limonene 0.20 0.20 0.31 0.31 0.26 0.26 Total 100 100 100 100 100 100

As a result of comparison of overall quantitative values of volatile aroma ingredients (alcohols, aldehydes, ketones, esters and acids), it could be confirmed that the lactic acid bacteria (isolated strain, reference strain)-applied breads contained great amounts of esters. Esters are aroma ingredients having soft and mild fragrance and it could be confirmed that the bread containing the isolated strain (L. sanfranciscensis 142) according to the present invention offered soft and mild flavor and taste.

In addition, it could be confirmed that the lactic acid bacteria (isolated strain, reference strain)-applied breads contained great amounts of acids.

However, it could be confirmed that only commercial yeast-applied bread contained great amounts of alcohols. Alcohols are aroma ingredients having light and strong fragrance and it could be confirmed that the commercial yeast-applied bread had worse flavor and taste spectrum.

FIG. 25 shows results of comparison of quantitative values of aroma ingredients of bread containing Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 isolated from natural sourdough.

Meanwhile, as a result of analysis of representative aroma ingredients, as can be seen from Table 21, the lactic acid bacteria (isolated strain, reference strain)-applied breads had a low content of ethyl alcohol having fresh fragrance, as compared to the control group (only commercial yeast-applied bread).

In addition, isoamyl alcohol having banana or European pear-like sweet flavor, 2-phenyl ethyl alcohol having corn-like sweet flavor, furfural having caramel, candy and almond flavors, acetoin of ketone having soft buttery taste, and esters having sweet flavor were found to be present in higher amounts in the lactic acid bacteria-applied breads. In addition, the reference strain-applied bread had a different acid composition from those of the control group and isolated strain-applied bread.

Example 14 Sensory Evaluation and Characterization of Breads Containing Saccharomyces cerevisiae (Sac. serevisiae), Lactobacillus curvatus (L. curvatus), Lactobacillus brevis (L. brevis), and Lactobacillus sanfranciscensis (L. sanfranciscensis) Isolated from Natural Sourdough

In the present example, sensory evaluation and characteristics of breads containing Saccharomyces cerevisiae (Sac. serevisiae), Lactobacillus curvatus (L. curvatus), Lactobacillus brevis (L. brevis), and/or Lactobacillus sanfranciscensis (L. sanfranciscensis) isolated from natural sourdough (Nuruk-containing Korean sourdough) were confirmed.

After tasting respective breads produced in the examples given above, sensory evaluation was conducted in terms of texture, flavor and/or taste. Results of sensory evaluation were confirmed on a scale of 1 to 9 with respect to texture (mouthfeel) and flavor and/or taste (9-considerably excellent, 1-considerably bad).

In addition, measurement methods of specific volume, pH, TTA and water content with respect to respective breads were the same as in Example 10 given above.

Results of sensory evaluation and characterization are shown in the following Table 22.

TABLE 22 Sensory evaluation and characteristics of applied products Reference Control Isolated Reference Reference strain Isolated group yeast strain Isolated strain Isolated (L. sanfran- strain (only (Sac. (L. curvatus strain (L. brevis strain ciscensis (L. sanfran- commercial serevisiae KCCM (L. curvatus KACC (L. brevis KACC ciscensis yeast- 01435)- 40715)- 104)- 11433)- 149)- 12431)- 142)- applied applied applied applied applied applied applied applied Items bread) bread bread bread bread bread bread bread Specific 4.92 4.98 4.98 5.01 5.12 5.15 5.01 5.03 volume *Texture 7.5 8.0 7.8 7.9 7.2 7.7 7.2 7.9 *Flavor 7.5 8.0 8.1 8.2 7.6 7.6 7.4 7.4 and/or taste pH 5.53 5.46 5.41 5.39 5.25 5.29 5.29 5.19 TTA 2.27/4.87 2.43/4.91 2.51/5.22 2.55/5.39 3.65/6.86 3.54/6.61 3.55/6.15 4.56/7.57 (6.6/8.5) Water 41.67 41.73 41.70 41.71 42.45 42.39 41.25 41.28 content (%)

As a result of testing, it could be seen that the isolated yeast (Sac. serevisiae 01435)-applied bread exhibited superior texture due to softness, had mild flavor and taste due to less gas odor, and gained superior texture and flavor and/or taste scores, as compared to the control group (commercial yeast)-applied bread.

Lactobacillus curvatus (L. curvatus 104) isolated strain-applied bread grained higher texture and flavor and/or taste scores as compared to the control group (commercial yeast-applied bread). There was no significant difference between the isolated strain and the reference strain (L. curvatus KCCM 40715).

It could be seen that the Lactobacillus brevis (L. brevis 149) isolated strain-applied bread grained higher texture and flavor and taste scores than the control group (commercial yeast) and reference strain (L. brevis KACC 11433). The isolated strain-applied breads were soft in texture, whereas the reference strain-applied bread was sticky or lumpy in texture and did not receive a good evaluation.

Lactobacillus sanfranciscensis (L. sanfranciscensis 142) isolated strain-applied bread had better texture than the control group (commercial yeast) and the reference strain (L. sanfranciscensis KACC 12431). The isolated strain-applied bread was soft in texture, whereas the reference strain-applied bread was sticky or lumpy in texture and thus did not receive a good evaluation.

Example 15 Application of Saccharomyces cerevisiae (S. serevisiae), Lactobacillus curvatus (L. curvatus), Lactobacillus brevis (L. brevis), and Lactobacillus sanfranciscensis (L. sanfranciscensis) Isolated from Natural Sourdough and a Combination Thereof to Bread Making

In the present example, Saccharomyces cerevisiae (S. serevisiae) 01435, a Lactobacillus curvatus (L. curvatus) 104 strain, a Lactobacillus brevis (L. brevis) 149 strain, and a Lactobacillus sanfranciscensis (L. sanfranciscensis) 142 strain isolated from natural sourdough (Nuruk-containing Korean sourdough) and a combination of the strains (S. serevisiae 01435, L. curvatus 104, L. brevis 149 and L. sanfranciscensis 142) were applied to bread making and characteristics thereof were compared.

(1) Production and Analysis of Dough Fermented with Lactic Acid Bacteria

Lactic acid bacteria-fermented dough were prepared using Lactobacillus curvatus (L. curvatus) 104, Lactobacillus brevis (L. brevis) 149, Lactobacillus sanfranciscensis (L. sanfranciscensis) 142, and a combination of Lactobacillus (L. curvatus 104, L. brevis 149 and L. sanfranciscensis 142) and characteristics thereof were analyzed.

100 g of a strong flour was mixed with 2×10¹⁰ cfu/g of lactic acid bacteria and 100 g of heating/cooling water, the mixture was fermented at 30° C. in a fermenter to prepare dough fermented with lactic acid bacteria, the dough was cooled when pH thereof reached 4.2±0.2, and pH, TTA and the number of bacteria of lactic acid bacteria-fermented dough were measured. At this time, pH and TTA were measured in the same manner as in Example 10.

Meanwhile, the lactic acid bacteria were obtained by culturing in MRS broth at a temperature of 30° C. for 22±2 hours, centrifuging and washing with physiological saline, and the dough was first inoculated with 1×10⁸ cfu of the bacteria per 1 g of the dough.

In addition, lactic acid bacteria-fermented dough containing the combination of isolated strains was prepared by mixing equivalent amounts of lactic acid bacteria-fermented dough respectively applying Lactobacillus curvatus (L. curvatus) 104, Lactobacillus brevis (L. brevis) 149, and Lactobacillus sanfranciscensis (L. sanfranciscensis) 142.

Test results are shown in the following Table 23.

TABLE 23 Number TTA (based of Isolated strain Fermentation on 15 g, mL) bacteria dough time pH pH 6.6 pH 8.5 (cfu/g) L. curvatus 104 3 4.38 4.27 5.81 2.1 × 10⁹ L. brevis 149 6 4.40 5.72 7.32 1.4 × 10⁹ L. 6 4.30 5.38 7.00 2.3 × 10⁹ sanfranciscensis 142 L. curvatus 104 + — 4.34 4.30 5.95 1.7 × 10⁹ L. brevis 149 + L. sanfranciscensis 142

As can be seen from Table 23, there was no significant difference in pH between respective dough, and the number of bacteria of Lactobacillus curvatus (L. curvatus) 104-applied dough was 2.1×10⁹ cfu/g, the number of bacteria of Lactobacillus brevis (L. brevis) 149-applied dough was 1.4×10⁹ cfu/g, the number of bacteria of Lactobacillus sanfranciscensis (L. sanfranciscensis) 142-applied dough was 2.3×10⁹cfu/g, and the number of bacteria of the combined isolated strain (L. curvatus 104, L. brevis 149, L. sanfranciscensis 142)-applied dough was 1.7×10⁹cfu/g.

(2) Bread Making

As can be seen from following Table 24, the isolated yeast and isolated strains were combined and respective groups were designated “E˜H sets”. Then, the sets were applied to breads and characteristics were confirmed according to composition.

Breads were made as follows. Ingredients (strong flour, yeast, Rimulsoft and purified water) constituting sponge dough as shown in the following Table 25 were put into a mixer (SK101S MIXER®, Japan), kneaded in a second stage for 2 minutes and in a third stage for 1 minute and then further mixed until the final temperature of the kneaded substance reached 25° C. Then, the mixture was allowed to stand at room temperature for 30 minutes and primarily fermented at 6° C. in a fermenter for 16 hours to prepare sponge dough.

Then, ingredients constituting dough (strong flour, refined salt, refined sugar, whole milk powder, yeast, purified water and lactic acid bacteria-fermented dough) as shown in the following Table 25 were put in a mixer (SK101S MIXER®, Japan), kneaded in a first stage for 1 minute, the sponge dough was added thereto and the resulting mixture was further mixed in a second stage for 3 minutes and in a third stage for 2 minutes. Then, butter was added to the mixture, and the resulting mixture was kneaded in the second stage for 3 minutes and in the third stage for 3 minutes until the final temperature of the kneaded substance reached 27° C. to prepare dough.

The dough was secondarily fermented in a fermenter at 27° C. and at relative humidity of 85% for 30 minutes, cut to a predetermined size, made round and aged in a fermenter at 27° C. and at relative humidity of 85% for 15 minutes. After aging, the dough was molded and put into a bread case. Then, the dough put into the bread case was fermented under the conditions of 37° C. and a relative humidity of 85% for 50 minutes to prepare bread dough. The bread dough was baked in an oven at an upper heat of 170° C. and at a lower heat of 210° C. for 35 minutes. Then, the bread was cooled at room temperature until an inner temperature thereof reached 32° C.

Images of the produced control group (only commercial yeast-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread, and Saccharomyces cerevisiae 01435, and Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread are shown in FIG. 26 (FIG. 26). FIG. 26 shows images of the control group (only commercial yeast-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread, Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread, and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread.

TABLE 24 Items Experimental group Control group Commercial yeast E set S. cerevisiae 01435 + L. curvatus 104 F set S. cerevisiae 01435 + L. brevis 149 G set S. cerevisiae 01435 + L. sanfranciscensis 142 H set S. cerevisiae 01435 + L. curvatus 104 + L. brevis 149 + L. sanfranciscensis 142

TABLE 25 Control Ingredients group E set F set G set H set Sponge Strong flour 70 70 70 70 70 dough Commercial 0.7 — — — — yeast Isolated yeast — 36 36 36 36 Rimulsoft 0.3 0.3 0.3 0.3 0.3 Feed water 42 42 42 42 42 Dough Strong flour 30 20 20 20 20 Refined salt 1.8 1.8 1.8 1.8 1.8 Refined sugar 7 7 7 7 7 Whole milk 3 3 3 3 3 powder Butter 10 10 10 10 10 Commercial 0.6 — — — — yeast Isolated yeast — 31 31 31 31 Feed water 23 13 13 13 13 Lactic acid — 20 20 20 20 bacteria- fermented dough (Unit: g)

(3) Measurement of Physical Properties of Bread

Physical properties (pH, total titratable acidity, chromaticity) of control group (only commercial yeast-applied bread), E set-applied bread, F set-applied bread, G set-applied bread and H set-applied bread were measured. Measurement of pH, total titratable acidity and chromaticity was carried out using the same method as in Example 10 and results are shown in the following Table 26.

TABLE 26 Control group (only commercial E set-applied F set-applied G set-applied H set-applied yeast-applied bread) bread bread bread bread pH 5.53 5.27 5.26 5.31 5.29 TTA (6.6/8.5) 2.27/4.87 3.37/5.24 3.41/5.31 3.21/5.13 3.29/5.21 Water 41.67% 41.42% 41.51% 41.49% 41.57% content Secondary 55 minutes 55 minutes 54 minutes 55 minutes 55 minutes fermentation time Specific volume 4.92 4.94 4.94 4.90 4.98 Hunter lab L 85.11 84.54 84.92 85.01 84.91 color values a −2.06 −2.31 −2.11 −2.18 −2.17 b 17.92 17.42 17.23 17.31 17.29

As a result of measurement, it could be seen that the isolated strain (E, F, G, H set)-applied breads had lower pH than the control group. In addition, it could be seen that the H set (S. cerevisiae 01435+L. curvatus 104+L. brevis 149+L. sanfranciscensis 142)-applied bread had slightly greater specific volume than other breads.

(4) Confirmation of Gas Generation Capability of Dough

Gas generation capability was compared between the control group (only commercial yeast-applied dough), E set-applied dough, F set-applied dough, G set-applied dough and H set-applied dough. The gas generation capability was measured on 25 g of dough using a gas generation capability measurement device (fermometer) at 30° C. for 10 hours.

As a result, E, F, G and H set-applied dough exhibited overall similar gas generation capability to the control group, whereas, in an early stage, E, F, G and H set-applied dough exhibited slightly superior gas generation capability (FIG. 27). FIG. 27 shows confirmation results of gas generation capability of only commercial yeast-applied dough (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied dough (E set-applied dough), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied dough (F set-applied dough), Saccharomyces cerevisiae 01435 and L. sanfranciscensis 142 strain-applied dough (G set-applied dough), and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and L. sanfranciscensis 142 strain-applied dough (H set-applied dough).

(5) Measurement of Aging Level of Bread

Hardness and aging rate over time were compared between control group (only commercial yeast-applied bread), E set-applied bread, F set-applied bread, G set-applied bread and H set-applied bread.

Hardness and aging rate over time were measured in the same manner as in Example 10, hardness values are shown in the following Table 27 and results of aging rate over time are shown in FIG. 28.

TABLE 27 Sample Hardness On the 1^(st) day (after 19 hours) Control group (only commercial yeast-applied bread) 165.972 E set (S. cerevisiae 01435 + L. curvatus 104)-applied 196.242 bread F set (S. cerevisiae 01435 + L. brevis 149)-applied 170.435 bread G set (S. cerevisiae 01435 + L. sanfranciscensis 142)- 197.895 applied bread H set (S. cerevisiae 01435 + L. curvatus 104 + 182.766 L. brevis 149 + L. sanfranciscensis 142)-applied bread On the 3^(rd) day (after 63 hours) Control group (only commercial yeast-applied bread) 260.353 E set (S. cerevisiae 01435 + L. curvatus 104)-applied 249.482 bread F set (S. cerevisiae 01435 + L. brevis 149)-applied 248.281 bread G set (S. cerevisiae 01435 + L. sanfranciscensis 142)- 238.799 applied bread H set (S. cerevisiae 01435 + L. curvatus 104 + 253.967 L. brevis 149 + L. sanfranciscensis 142)-applied bread On the 4^(th) day (after 87 hours) Control group (only commercial yeast-applied bread) 281.774 E set (S. cerevisiae 01435 + L. curvatus 104)-applied 269.967 bread F set (S. cerevisiae 01435 + L. brevis 149)-applied 275.289 bread G set (S. cerevisiae 01435 + L. sanfranciscensis 142)- 264.063 applied bread H set (S. cerevisiae 01435 + L. curvatus 104 + 268.063 L. brevis 149 + L. sanfranciscensis 142)-applied bread

As a result, E, F, G and H set-applied breads had lower hardness and were thus softer than the control group (only commercial yeast-applied bread).

Meanwhile, as a result of analysis of aging rate over time, all breads exhibited similar aging rate (FIG. 28). FIG. 28 shows confirmation results of aging rates of only commercial yeast-applied bread (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread (E set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread (F set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread (G set-applied bread), and Saccharomyces cerevisiae 01435, and Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread (H set-applied bread).

(6) Analysis of Aroma Ingredients of Bread

Aroma ingredients were analyzed using a GC/MS system to compare expression of flavor and taste ingredients between the control group (only commercial yeast-applied bread) and E, F, G, H set-applied breads.

Analysis was conducted on 1 g of a sample and GC/MS analysis conditions were the same as in Table 5 given above. After GC/MS analysis, the overall quantitative values of alcohols, aldehydes, ketones, esters and acids were compared (FIG. 29), and relative proportions of 22 types of respective representative aroma ingredients are shown as percentages in the following Table 28.

TABLE 28 Content proportion (%) of aroma ingredients Content Content Content Content contained in proportion proportion proportion proportion control group (%) of aroma (%) of aroma (%) of aroma (%) of aroma (only ingredients ingredients ingredients ingredients commercial contained in E contained in F contained in G contained in H yeast-applied set-applied set-applied set-applied set-applied Aroma ingredients bread) bread bread bread bread Alcohol Ethyl alcohol 55.53 87.38 43.87 86.79 41.01 83.80 43.17 83.49 48.32 86.26 1-Propanol 0.52 0.39 0.47 0.48 0.40 2-Methyl-1- 0.31 3.05 2.51 2.47 2.79 propanol Isoamyl alcohol 18.05 21.16 19.70 18.77 19.40 1-Hexanol 1.24 1.48 1.48 1.64 1.50 2-Phenyl 11.72 16.85 18.63 16.96 13.85 ethyl alcohol Aldehyde Hexanal 0.64 3.45 0.56 3.95 0.67 3.70 0.72 3.89 0.65 3.50 Nonanal 0.82 0.87 1.35 1.26 0.81 Furfural 0.60 0.89 0.57 0.70 0.64 Benzaldehyde 1.38 1.63 1.11 1.22 1.40 Ketone 2-Heptanone 0.47 2.33 0.48 3.65 0.57 4.38 0.59 4.80 0.80 4.25 Acetoin 1.33 2.54 2.85 3.27 2.86 2-Nonanone 0.54 0.63 0.96 0.93 0.59 Ester Ethyl hexanoate 1.31 6.18 1.61 4.63 1.73 5.90 1.68 5.70 1.33 4.54 Ethyl octanoate 3.81 2.39 3.38 3.15 2.17 Ethyl decanoate 0.42 0.27 0.35 0.33 0.22 Isoamyl lactate 0.64 0.35 0.44 0.54 0.82 Acid Octanoic acid 0.42 0.42 0.00 0.80 0.42 1.99 0.21 1.84 0.00 1.15 Acetic acid 0.00 0.25 0.48 0.51 0.54 Hexanoic acid 0.00 0.55 1.09 1.12 0.60 Others Alpha-limonene 0.24 0.24 0.18 0.18 0.24 0.24 0.29 0.29 0.30 0.30 Total 100 100 100 100 100 100 100 100 100 100

As a result of comparison of overall quantitative values of volatile aroma ingredients (alcohols, aldehydes, ketones, esters and acids), it could be confirmed that E, F, G, and H set-applied breads contained higher amounts of Ketones having soft and mild fragrance than the control group (only commercial yeast-applied bread), in particular, the H set-applied bread contained the highest amount of ketone. In addition, aldehydes having soft and mild flavor were found to be present in a high amount in the E set-applied bread. In addition, it could be confirmed that E, F, G and H set-applied breads contained greater amounts of acids than the control group-applied bread.

However, the control group contained higher amounts of esters having light and strong fragrance and thus had a worse flavor and taste spectrum.

FIG. 29 shows results of comparison of quantitative values of aroma ingredients of only commercial yeast-applied bread (control), Saccharomyces cerevisiae 01435 and Lactobacillus curvatus 104 strain-applied bread (E set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus brevis 149 strain-applied bread (F set-applied bread), Saccharomyces cerevisiae 01435 and Lactobacillus sanfranciscensis 142 strain-applied bread (G set-applied bread), and Saccharomyces cerevisiae 01435, Lactobacillus curvatus 104 strain, Lactobacillus brevis 149 strain and Lactobacillus sanfranciscensis 142 strain-applied bread (H set-applied bread).

Meanwhile, as a result of analysis of representative aroma ingredients, as can be seen from Table 28 given above, E, F, G, H set-applied breads had a low content of ethyl alcohol having fresh flavor as compared to the control group (only commercial yeast-applied bread). In addition, isoamyl alcohol having banana or European pear-like sweet flavor and 2-phenyl ethyl alcohol having corn-like sweet flavor were found to be present in higher amounts in E, F, G, and H set-applied breads, and nonanal having citrus and fatty flavors (pleasant flavor) was found to be present in higher amounts in F and G set-applied breads. In addition, regarding acetoin of ketone having soft buttery taste, E, F, G, H set-applied breads had overall higher contents than the control group. In addition, E, F, G, H set-applied breads had overall higher acid contents, in particular, F and G set-applied breads had remarkably higher acid contents than the control group.

Example 16 Sensory Evaluation and Characterization of Breads Containing Saccharomyces cerevisiae (S. serevisiae), Lactobacillus curvatus (L. curvatus), Lactobacillus brevis (L. brevis), and/or Lactobacillus sanfranciscensis (L. sanfranciscensis) Isolated from Natural Sourdough, or a Combination Thereof

In the present example, sensory evaluation and characteristics of the control group (only commercial yeast-applied bread), E set (S. cerevisiae 01435+L. curvatus 104)-applied bread, F set (S. cerevisiae 01435+L. brevis 149)-applied bread, G set (S. cerevisiae 01435+L. sanfranciscensis 142)-applied bread, and H set (S. cerevisiae 01435+L. curvatus 104+L. brevis 149+L. sanfranciscensis 142)-applied bread were confirmed.

After tasting respective breads produced in the examples given above, sensory evaluation was conducted in terms of texture and flavor and/or taste. Results of sensory evaluation were confirmed on a scale of 1 to 9 with respect to texture and flavor and taste (9-considerably excellent, 1-considerably bad).

In addition, measurement methods of specific volume, pH, TTA and water content with respect to respective breads were the same as in Example 10 given above.

Results of sensory evaluation and characterization are shown in the following Table 29.

TABLE 29 Sensory evaluation and characteristics of applied products H set (S. cerevisiae Control E set (S. F set (S. 01435 + group cerevisiae cerevisiae G set (S. L. curvatus (only 01435 + L. 01435 + cerevisiae 104 + L. brevis commercial curvatus L. brevis 01435 + L. 149 + L. yeast- 104)- 149)- sanfranciscensis sanfranciscensis applied applied applied 142)-applied 142)-applied Items bread) bread bread bread bread Specific volume 4.92 4.94 4.94 4.90 4.98 *Texture 7.5  7.8  7.4  7.4  7.5  *Flavor and/or 7.5  7.9  7.5  7.5  7.6  taste pH 5.53 5.27 5.26 5.31 5.29 TTA (6.6/8.5) 2.27/4.87 3.37/5.24 3.41/5.31 3.21/5.13 3.29/5.21 Water content  41.67%  41.42%  41.51%  41.49%  41.57% (%)

As a result of testing, it could be seen that the isolated yeast and isolated strain (E, F, G and H set)-applied breads were soft and thus exhibited superior texture, and emitted less gas odor and were thus mild in flavor and taste, and gained superior texture and flavor and/or taste scores, as compared to the control group (commercial yeast)-applied bread. 

1. Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP).
 2. Dough for baking produced by adding Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) to flour, followed by fermentation.
 3. The dough for baking according to claim 2, wherein the dough for baking is produced by further adding one or more selected from Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP), Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) to flour, followed by fermentation.
 4. A bread produced by adding Saccharomyces cerevisiae SPC-SNU 70-1 (KCTC 12776BP) to flour, followed by fermentation and baking.
 5. The bread according to claim 4 wherein the bread is produced by further adding one or more selected from Lactobacillus curvatus SPC-SNU 70-3 (KCTC 12778BP), Lactobacillus brevis SPC-SNU 70-2 (KCTC 12777BP) and Lactobacillus sanfranciscensis SPC-SNU 70-4 (KCTC 12779BP) to flour, followed by fermentation and baking. 